Browsing by Author "Sipahioglu, Hikmet Murat"

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Bougainvillea Spectabilis Willd. Bitkisinin Biyolojik Olarak Aktif Rekombinant Bouganin Proteininin Antiviral Ve Antifungal Aktivitesi
(2018) Durak, Emre Demirer; Sipahioglu, Hikmet Murat; Usta, Mustafa; Güller, Abdullah
Bougainvillea spectabilis Willd. bitkisinden ribozom inaktive eden proteinlerden olan Bouganin antiviral protein (BAP) geni izole edilerek klonlanmış, ifade edilmiş, antiviral ve antifungal özellikleri araştırılmıştır. Bouganin proteinini kodlayan genin tamamı bitkinin olgun yapraklarından ekstrakte edilen mRNA’dan Reverse Transkripsiyon-PCR yöntemi ile çoğaltılmıştır. Gen, uçtan uca bölgeyi kapsayacak şekilde tasarlanan spesifik primerler yardımı ile prokaryotik ekspresyon vektörü pETDuet-1’de klonlanmıştır. BAP geninin ifadesi rekombinant plazmitin Escherichia coli (BL21(DE3)pLysS) hücrelerine transferi sonrası isopropyl β-D thiogalactopyranoside (IPTG) ile teşvik edilmiştir. Molekül ağırlığı yaklaşık 28 kDa olan BAP transforme edilmiş bakterilerden izole edilmiştir. BAP ‘ın antiviral aktivitesi Zucchini yellow mosaic virus (ZYMV) kullanılarak mekanik inokulasyon yöntemi ile araştırılmıştır. Antifungal aktivitenin belirlenmesi disk difüzyon metodu yardımıyla patojen ve patojen olmayan Rhizoctonia solani, Trichoderma harzianum ve Fusarium oxysporum fungusları ile araştırılmıştır. BAP uygulama miktarı arttıkça ZYMV’nin neden olduğu hastalık şiddetinin azaldığı tespit edilmiştir. BAP 72 saatlik kontrol uygulaması ile karşılaştırıldığında, Rhizoctonia solani’nin gelişimini % 30.7, Trichoderma harzianum’unun gelişimini ise % 20 oranında inhibe etmiştir. Fusarium oxysporum’un gelişiminde herhangi bir inhibisyon etkisi gözlenmemiştir. In vitro’da ifade edilen BAP proteininin uygulandığı tüm bitkiler (sadece BAP protieni uygulanan kontrol grubu da dahil) uygulama yapılmayan bitkilere oranla şiddetli gelişme geriliği sergilemiştir.
Cloning and Sequencing of Coat Protein Gene of Zucchini Yellow Mosaic Virus Isolated From Squash and Muskmelon in Turkey
(Tubitak Scientific & Technological Research Council Turkey, 2012) Ozer, Meryem; Sipahioglu, Hikmet Murat; Usta, Mustafa; Fidan, Hakan
The coat protein (CP) genes of the genomic RNA of 2 severe Turkish isolates of Zucchini yellow mosaic virus (ZYMV) from squash and muskmelon [ZYMV-Adana (Ad) and ZYMV-Ahlat (Ah), respectively] were cloned, and their complete nucleotide sequences and deduced amino acids were determined. The analysis revealed that both Turkish ZYMV-CP genes contained 837 nucleotides encoded for a CP of about 31.2 kDa. Phylogenetic trees based on nucleic acid sequences were constructed by the neighbor joining and unweighted pair group mean arithmetic (UPGMA) methods with 100 bootstrap replicates. A high degree of homology was detected between the 2 Turkish isolates on the nucleotide sequences (97%). The CP sequence of ZYMV-Ad and ZYMV-Ah varied among the 23 isolates with overall identity of 93%-98% and 94%-99%, respectively, at the nucleotide level. Comparison of the nucleotide sequences of 23 isolates from different geographical regions worldwide showed the ZYMV-Ad isolate clustered with isolates from Middle Eastern countries (Israel, Jordan, and Syria); ZYMV-Ah isolate was clustered with isolates from Far Eastern countries (Korea and Taiwan). The N-terminal of the Turkish ZYMV-Ah CP contained a distinctive sequence at nucleotide positions 9324-9328, which distinguished the Turkish ZYMV-Ah isolate from all previously reported ZYMV isolates. The CP cistron of the ZYMV-Ad and Ah isolates contained 279 amino acid residues. Pairwise nucleotide sequence comparison revealed sequence similarities of 58%-70% between ZYMV Turkish isolates and 22 other potyviruses. Mechanical inoculations showed that ZYMV-Ah produced faster systemic symptom induction than ZYMV-Ad on squash, suggesting that ZYMV-Ah was a more severe isolate than ZYMV-Ad (GenBank accession JF317296-JF317297).
Detection, in Silico Analysis and Molecular Diversity of Phytoplasmas From Solanaceous Crops in Turkey
(Czech Academy Agricultural Sciences, 2022) Usta, Mustafa; Guller, Abdullah; Sipahioglu, Hikmet Murat
Phytoplasma-like symptoms of leaf yellowing and calyx malformation were observed in eggplant (Solanum melongena L.), upward leaves and fruit malformation in pepper (Capsicum annuum L.), and aerial tuber formation in potato (S. tuberosum L.) during the survey performed in the late season (August to September) of 2015 and 2016 in Van province (Turkey). A total of 100 samples were tested by nested-PCR using universal primer pairs to assess the sanitary status of the solanaceous crops and to characterise the phytoplasma isolates. Among them, seven sam-ples resulted in a 1.25 kb DNA fragment, and five (two eggplants, two peppers, and one potato) were molecularly characterised (Accession No.: KY579357, KT595210, MF564267, MF564266, and MH683601). BLAST and the virtual restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed the presence of two distinct phytoplasma infections in solanaceous crops: `Candidatus Phytoplasma trifolii' a member of the clover proliferation group (16SrVI) and subgroup A and `Candidatus P. solani' a member of the stolbur group (16SrXII) and subgroup A. The virtual RFLP analysis and calculated coefficients of RFLP pattern similarities further revealed a remarkable ge-netic diversity among the `Candidatus P. solani' isolates infecting pepper (similarity coefficient of 0.90) and eggplant (similarity coefficients of 0.98 and 1.00) at the same geographical area. This is the first report of the natural occurrence of `Candidadtus P. trifolii' in potato from the Eastern Anatolia region, Turkey.
First Report of "candidatus Phytoplasma Solani" on a New Host Marigold (Tagetes Erecta L.)
(Tubitak Scientific & Technological Research Council Turkey, 2016) Alp, Sevket; Usta, Mustafa; Sipahioglu, Hikmet Murat; Guller, Abdullah
Marigold (Tagetes erecta L.) plants, also called Mexican or Aztec marigold, with symptoms of shoot proliferation, dwarfing, and reddening were observed in ornamental gardens of Van Province (Turkey). Five plants, two of them showing reddening and three symptomless plants, were sampled at the end of September 2014. Genomic DNA isolated from symptomatic and nonsymptomatic plant leaves was used to amplify 16S rDNA fragments by nested polymerase chain reaction (PCR). Of the 5 marigold samples tested by PCR, only the two showing reddening symptoms yielded the expected 1.2-kb DNA fragments. Amplified PCR fragments were cloned into a plasmid vector and transformed into competent Escherichia coli strain JM 109. Recombinant plasmid DNA was isolated and sequenced bidirectionally. The provided sequences were 1244 bp and 1245 bp in length and were designated as isolate 1 and isolate 2, respectively. BLAST analysis of the 16S rDNA sequence and virtual restriction fragment length polymorphism (RFLP) analysis confirmed the presence of the phytoplasma "Candidatus Phytoplasma solani". The in silico virtual RFLP pattern of isolate 1, based on the 16S rDNA F2n/R2 fragment, was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group XII, subgroup A (GenBank accession no.: AF248959). Isolate 1 was identified as a member of 16SrXII-A. Based on the same analyses, isolate 2 showed molecular characteristics different from reference patterns of all previously established 16Sr groups and subgroups. The most similar was the reference pattern of 16Sr group XII, subgroup A (GenBank accession no.: AF248959), with a similarity coefficient of 0.97. This is the first report of naturally occurring "Ca. P. solani" affecting T. erecta, which shows that this plant species is an alternate host of the agent.
First Report of 'candidatus Phytoplasma Trifolii' Associated With Leaf Reddening and Upright Growth in Pears (Pyrus Communis L.)
(Czech Academy Agricultural Sciences, 2021) Usta, Mustafa; Guller, Abdullah; Sipahioglu, Hikmet Murat
The natural occurrence of 'Candidatus Phytoplasma trifolii' in pear trees (Pyrus communis Linnaeus) is reported here for the first time. In 2017, a total of thirty-five pear trees, two of them exhibiting leaf rolling along the midvein, reddening, bushy appearance, and upright growth symptoms were sampled in different locations in Van province, Turkey. The total deoxyribonucleic acid was extracted from symptomatic and asymptomatic plants. The purified DNA served as a template in nested polymerase chain reaction (nested-PCR) assays, performed to amplify 16S rRNA sequences using universal primer pairs (R16mF2/R16mR1 and R16F2n/R16R2). The resulting PCR products were then cloned into a pGEM T-Easy vector and sequenced bidirectionally. The phytoplasma strain, group, and subgroup identity were determined using the in silico restriction fragment length polymorphism (RFLP) analysis of the 16S ribosomal RNA-encoding gene sequences profiling with seventeen distinct restriction enzymes. Of the thirty-five pear samples, only two yielded 1 256 bp and 1 258 bp DNA fragments and were designated as Van-Pr3 (Acc. No. MH709141) and Van-Pr4 (Acc. No. MH730561), respectively. Based on the in silico virtual RFLP pattern analysis of the 16S rRNA sequences, we confirmed the presence of 'Ca. P. trifolii' belonging to the clover proliferation group and both identified phytoplasmas were identical with the similarity coefficient of 1.00 to the reference pattern of 16Sr group VI, subgroup A (Acc. No. AY390261). Here we report that the pear tree is an alternate host of the 'Ca. P. trifolii'.
Hıyar (Cucumis Sativus L.) Bitkisinde Aynı Simptomatolojiye Sahip İki Farklı Fitoplazma Etmeninin 16s Rdna'sının Rflp ve Nükleotid Dizi Analizleri ile Teşhisi ve Karakterizasyonu
(2017) Usta, Mustafa; Güller, Abdullah; Sipahioglu, Hikmet Murat
Van ilinde yetiştiriciliği yapılan hıyarlarda (Cucumis sativus L.) fitoplazma benzeri belirtiler görülmüştür. Gözlenen en önemli belirtiler arasında şiddetli cüceleşme, cadı süpürgesi görünümü, rozetleme ve küçük yapraklılık yer almaktadır. Toplanan 8 hıyar örneğine R16mF2/R16mR1 ve R16F2n/R16R2 primer çiftleri ile uygulanan Nested-PCR testi uygulanmıştır. Belirti gösteren ve göstermeyen hıyar yapraklarından izole edilen genomik DNA'ların kullanıldığı PCR reaksiyonunda fitoplazma etmenlerine ait 16S rDNA fragmentleri çoğaltılmış ve test edilen sekiz örneğin dördünde 1.25 kb uzunluğunda DNA fragmentleri elde edilmiştir. Beklenen band büyüklüğünü veren örneklerden rastgele seçilen iki örnek uygun bir plasmid vektörde klonlanmıştır. Saflaştırılan rekombinant plasmid DNA'sı çift yönlü olarak dizilenmiş ve 16S rDNA dizisinin BLAST ve RFLP analizleri yapılmıştır. Şiddetli fitoplazma belirtisi gösteren hıyar numunelerinin birinde 'Candidatus Phytoplasma solani' (benzerlik katsayısı 1.00) (GenBank erişim no: KX977570), diğerinde ise 'Candidatus Phytoplasma trifolii' (benzerlik katsayısı 0.98) (GenBank erişim no: KR080212) saptanmıştır. İzolatlardan birincisine'Van-solani' diğerine ise 'Vantrifolii' izolatı isimleri verilmiştir. İki farklı fitoplazma etmeninin oluşturduğu belirtiler arasında bir fark gözlenmemiştir. Yazarların bilgisine göre, Türkiye'de hıyarları doğal olarak enfekte eden \"Ca. P. solani\" ve \"Ca. P. trifolii\" fitoplazma etmenleri ilk defa bu çalışma ile rapor edilmiştir
Incidence and Genetic Stability of Potato Spindle Tuber Pospiviroid in Potato in Turkey
(Tubitak Scientific & Technological Research Council Turkey, 2012) Guner, Uftade; Sipahioglu, Hikmet Murat; Usta, Mustafa
The prevalence of Potato spindle tuber pospiviroid (PSTVd) infection in randomly selected potato tubers was determined via nonisotopic dot blot hybridization and reverse transcriptase polymerase chain reaction (RT-PCR). In January and February of 2010, 168 seedling tubers of Solanum tuberosum, representing 27 cultivars, were received from different seedling potato producers in Turkey. Digoxigenin-labeled RNA probes of PSTVd were synthesized from the cloned PCR products and applied in dot blot hybridization to detect viroids in RNA extracts isolated from dormant potato tubers. PSTVd was found in 7 samples and 6 cultivars (Innovator, Lady Jo, Russet Burbank, Agria, Provento and Konsul). Some RNA samples, although positive by RT-PCR test, did not give a hybridization signal by dot blot assay. The complete genome of a selected isolate (PSTVd-TR) was cloned and sequenced. Primary and secondary structure analysis showed that the RNA genome of the PSTVd-TR isolate (GenBank Accession No. HQ456944) differed from other compared isolates by only a few nucleotides. The highest identity (99%) was in a PSTVd isolate that was identified in Solanum spp. in Italy (Accession No. EF459700). The infectivity of PSTVd-TR isolate was shown 2 weeks after the mechanical inoculation of sap extracts onto seedlings of tomato cultivar Joker. The PSTVd infections were proven by RT-PCR assay in symptomless tomato seedlings.
Molecular Analysis of 'candidatus Phytoplasma Trifolii' and 'candidatus Phytoplasma Solani' Associated With Phytoplasma Diseases of Tomato (Pdt) in Turkey
(Friends Science Publ, 2018) Usta, Mustafa; Guller, Abdullah; Sipahioglu, Hikmet Murat
Tomato plants displaying severe fruit deformation, flower sterility, aerial rooting, purplish leaves and leaf rolling were observed in tomato fields at Van province (Turkey). Samples were collected, and total DNA was extracted from symptomatic and asymptomatic plants. Nested polymerase chain reaction (nested-PCR) assays were performed to amplify 16S rDNA sequences for molecular detection using universal primer pairs. Out of 100 tested tomato samples, 11% of tomato samples yielded a DNA fragment of 1.25 kb. Amplified PCR products were then cloned into pGEM T-Easy vector and sequenced using new generation DNA sequencing (NGS) system. The virtual restriction fragment length polymorphism (RFLP) analysis of 16S rDNA sequences and molecular detections were allowed to characterize possible phytoplasmas associated with diseased plants. Our results revealed the presence of two Phytoplasma species belonging to two different ribosomal groups; 'Candidatus Phytoplasma trifolii' (16Sr VI-A group) (Acces no. MF564268, MG732925) and 'Candidatus Phytoplasma solani' (16SrXII-A group) (Acces no. KY579358, MF576263). Despite a high variation in their similarity coefficient of'Ca. P. solani' VTS2 (0.91) and 'Ca. P. trifolii' VTT1 (0.88) isolates, the infected tomato plants generally displayed similar disease symptoms during field observations. Due to its commercial interest, co-existing of these phytoplasmas in tomato fields is of great phytosanitary significance not only for tomato plants but also for other crops such as vegetables, ornamentals and field crops. With this study, 'Ca. P. trifolii' associated with phytoplasma diseases of tomato (PDT) has been reported for the first time in tomato in Turkey. (C) 2018 Friends Science Publishers
Molecular Cloning and Sequence Analysis of the Its Region of Nuclear Ribosomal Dna for Species Identification in Dodders (Cuscuta; Convolvulaceae)
(Friends Science Publ, 2017) Keskin, Fatma; Kaya, Ilhan; Usta, Mustafa; Demir, Ibrahim; Sipahioglu, Hikmet Murat; Nemli, Yildiz
Dodder (Cuscuta sp.) is an obligate parasitic plant that is very difficult to control. In plants the internal transcribed spacer (ITS) region of the 18S-5.8S-26S nuclear ribosomal DNA (nrDNA) has been considered one of the most important sequences for phylogenetic analysis. Here we report the analysis of nrDNA's ITS sequences as an efficient tool to study the phylogeny of dodders collected from various provinces of Eastern Anatolia (Turkey). Genomic DNA of six dodder samples belonging to 4 distinct species was extracted from body tissue samples. The sequences of 18S rRNA, ITS-1, 5.8S rRNA, ITS-2 and 26S rRNA regions of 4 Cuscuta species were determined by molecular cloning and sequencing. The identity of cloned fragments was compared to determine sequence identity using NCBI database. Bootstrap analysis of nrDNA of C. approximate, C. lupuliformis, C. campestris and C. babylonica indicated high sequence identity with similar sequences belonging to different geographical origins of the world retrieved from NCBI database. Our results clearly showed that the most stable secondary structure derived from the sequences obtained by universal ITS4 and ITS5 primers is very efficient tool for identification of Cuscuta species when used in combination with phylogenetic analysis. (C) 2017 Friends Science Publishers
Pokeweed (Phytolacca Americana L.) Antiviral Protein Inhibits Zucchini Yellow Mosaic Virus Infection in a Dose-Dependent Manner in Squash Plants
(Tubitak Scientific & Technological Research Council Turkey, 2017) Sipahioglu, Hikmet Murat; Kaya, Ilhan; Usta, Mustafa; Unal, Murat; Ozcan, Dilek; Ozer, Meryem; Pallas, Vicente
Pokeweed antiviral protein (PAP) of Phytolacca americana L. (pokeweed) is a single-chain ribosome-inactivating protein (RIP) characterized by its ability to depurinate plant ribosomes. Here, we isolated, cloned, and expressed the ribosome inactivating protein (RIP) gene, designated as pokeweed antiviral protein type 1 (PAP I), from the summer leaves of pokeweed collected from the Black Sea region (Turkey). Our findings presented here provide direct evidence that exogenous application of PAP I causes concentration-dependent inhibition of Zucchini yellow mosaic virus (ZYMV) infection on squash plants. Squash plants were exposed to PAP I protein with and without DMSO for four consecutive days. Regular spraying of approximately 30 kDa recombinant PAP I at 2 mu g mL(-1) concentration prevented treated plants from mechanical virus infection. PAP I showed antiviral activity in 9 plants out of 15 inoculated plants. Remarkably, simultaneous application of PAP, DMSO, and ZYMV did not prevent virus infection, suggesting that PAP did not have any effect on viral RNA. In the absence of ZYMV the purified peptide was not cytotoxic for squash plants, although a reduction of plant size, possibly caused by host ribosome depurination, was observed.
Prunus Yapraklarında Prunus Necrotic Ring Spot (Pnrsv) ve Apple Chlorotic Leaf Spot (Aclsv) Virüslerinin Dağılımı
(2005) Usta, Mustafa; Sipahioglu, Hikmet Murat; Ocak, Mustafa; Şavur, Orçun Burak; Polat, Bülent
Prunus necrotic ringspot virüs (PNRSV)'ü ile infekteli Prunus mahaleb ve Apple chlorotic leafspot virüs (ACLSV)'ü ile infekteli şeftali (P. persica L.) yapraklarının farklı bölgelerinden alınan doku diskleri bu virüslerin yaprak dokusundaki dağılımlarını belirlemek amacı ile sırasıyla enzyme-linked immunosorbent assay (ELISA) ve reverse transcriptase polymerase chain reaction (RT-PCR) teknikleri ile analiz edilmişlerdir. Gerçekleştirilen ELISA testleri sonucunda her iki virüsünde yaprak ayasında yaprak sapı bölgesinde daha konsantre oldukları ve konukçu yapraklarında düzensiz bir dağılım gösterdikleri tespit edilmiştir. Aynı yaprak bölgelerinin kullanıldığı RT-PCR testlerinde 'ise her iki virüsün genetik materyalinin tüm yaprak bölgeleri için birbirine yakın ölçülerde amplifikasyon ürünleri oluşturduğu belirlenmiş ve testlenen yaprak bölgeleri arasında viral konsantrasyon bakımından bariz farklılıkların olmadığı saptanmıştır. RT-PCR testi sonuçlarından elde edilen kesin, net ve dengeli teşhisi ifade eden bantlar, ACLSV ve PNRSV virüslerinin Prunus yapraklarının testlenen tüm bölgelerinde homojen bir dağılım sergilediğini göstermiştir. Her iki virüs, kullanılan test yöntemine göre konukçularında farklı dağılım sergilemişlerdir. Elde edilen bulgular ışığında PNRSV ile ACLSV'nin konukçularındaki dağılımını belirlemede ELISA testi ile PCR testi arasında bir korelasyon saptanmamıştır.
Simultaneous Production of Alpha and Beta Amylase Enzymes Using Separate Gene Bearing Recombinant Vectors in the Same Escherichia Coli Cells
(Tubitak Scientific & Technological Research Council Turkey, 2020) Ozcan, Dilek; Sipahioglu, Hikmet Murat
The present study describes the simultaneous expression of thermostable industrial alpha (alpha) and beta (beta) amylase enzymes that have been used widely in starch industry. Genomic DNA of Bacillus stearothermophilus DSM 22 strain for alpha amylase and, Thermoanaerobacterium (Clostridium) thermosulfurogenes DSM 2229 strain for beta amylase were used as gene sources. Both genes were ligated into pETDuet-1 expression vector separately and resulting recombinant vectors were transformed into Escherichia coli BL21 competent cells by electroporation. The cells were first transformed by pETDuet-1/ alpha Amy recombinant plasmid, then the competent cells carrying this plasmid were prepared for the transformation of pETDuet-1/beta Amy plasmid. Enzymatic activities of bacterial colonies were detected on LB agar staining with iodide. Both enzymes were more produced by IPTG induction in BL21 cells and were purified using Ni-NTA agarose column. SDS-PAGE and western blot analyses showed that the molecular weight of purified alpha and beta amylase to be approximately 60 kDa and 55kDa, respectively. The concentration of the purified alpha and beta amylase were calculated as 4.59 mu g/mL and 3.17 mu g/mL with IPTG as an inducer in LB medium. The present study proposes a novel and efficient method for the production of thermostable alpha and beta amylases at the same E coli cells containing separate engineered plasmid vectors.
Survey of Apple Mosaic Virus in Apple-Growing Provinces of East Anatolia (Malatya and Van) by Rna Probe Hybridization Assay and Rt-Pcr
(Tubitak Scientific & Technological Research Council Turkey, 2013) Korkmaz, Gulustan; Sipahioglu, Hikmet Murat; Usta, Mustafa
Two main apple growing provinces (Van and Malatya) of East Anatolia were surveyed for the presence of the Apple mosaic virus (ApMV). Dot-blot hybridization and RT-PCR tests were implemented to investigate the incidence of ApMV, testing a total of 481 samples collected from commercial apple orchards. Digoxigenin-labeled RNA probes of the ApMV were synthesized from the cloned PCR products and applied in dot-blot hybridization to detect the virus in RNA extracts isolated from fresh leaf tissues. A validated RNA probe (dot-blot) hybridization method was adopted to investigate the presence of ApMV infections in the apple orchards of East Anatolia. In order to determine the most suitable mass extraction methods and to ascertain the presence of virus, 3 RNA preparation procedures (QIAGEN RNA extraction kit, silica capture, and citric buffer) were tested. Among the tested extraction methods, silica capture was determined as the most suitable extraction method for dot-blot hybridization assays. ApMV was found in the surveyed locations with an average incidence of 0.8%. The infected trees showed apparent disease symptoms on the leaves of apple trees. The coat protein (CP) genes of 2 viral isolates selected from Malatya (ApMV-G and ApMV-M) were cloned and their complete nucleotide sequences and deduced amino acids were determined (GenBank acc. nos. JX155668 and JX155669). The CP cistron of the ApMV-G and M isolates contained 224 amino acid residues. Phylogenetic trees based on nucleic acid sequences were constructed by the neighbor-joining and the unweighted pair-group mean arithmetic methods with 100 bootstrap replicates. The CP sequence of ApMV-G and ApMV-M varied among the 21 isolates, with overall identity ranging from 88% to 99% and ranging from 91% to 99% at the nucleotide level.
Use of Dried High-Phenolic Laden Host Leaves for Virus and Viroid Preservation and Detection by Pcr Methods
(Elsevier, 2006) Sipahioglu, Hikmet Murat; Usta, Mustafa; Ocak, Mustafa
The efficiency of RNA extraction for Apricot latent virus (ApLV), Plum bark necrosis stem pitting associated virus (PBNSPaV), Prunus necrotic ring spot virus (PNRSV), Potato virus Y (PVY), and Apple scar skin viroid (ASSVd) from infected hosts is of great importance for molecular diagnosis by the polymerase chain reaction (PCR). A method is described for drying tissue to overcome phenolic inhibitors of viral RNA. This study showed that the infected host leaves, dried at 65 degrees C for 2 days and conserved at 4 degrees C in air proof conditions, serve as good sources for detection of viral and viroid pathogens by PCR methods. Preliminary results suggest that ApLV, PNRSV, PVY, and ASSVd were detected easily by reverse transcriptase-polymerase chain reaction (RT-PCR) and PBNSPaV by nested-RT-PCR with high amplification yields. No significant difference was observed between ethidium bromide-stained band profiles of dried compared to fresh leaves of infected samples. The RNA derived from dry leaf samples was suitable for detection studies. This simple and inexpensive method has proved very effective for long term conservation of virus and viroid isolates. (c) 2006 Elsevier B.V. All rights reserved.